Ischemic injury is a debilitating outcome of natural illness and is a complication of commonly performed medical procedures. Given the prevalence and severity of outcomes in ischemic injury, there is significant interest in developing better pharmacological and procedural approaches to improve patient outcomes. One approach that has shown significant promise in the laboratory setting, particularly in the context of planned medical procedures, is the use of delayed anesthetic preconditioning. Delayed anesthetic preconditioning is a phenomenon whereby a prior exposure to clinical concentrations of commonly used inhaled anesthetics (including isoflurane) induces the production of endogenous protective proteins. These protective proteins provide robust protection against subsequent ischemic insults in a variety of tissues including kidney, liver, heart and brain. Although many aspects of delayed anesthetic preconditioning have been described, a complete understanding of preconditioning mechanism has yet to emerge. Recent work from my lab identified metallothionein I + II genes (MT-I + II) as among those significantly regulated in rat liver, kidney and heart following a 2% isoflurane exposure in vivo (Edmands et al., 2005). Furthermore, Carmel et al., (2004) found MT-I + II mRNAs were strongly and rapidly up-regulated in response to ischemic preconditioning of rat spinal cord. Metallothioneins I + II are small (6-7kDaltons), cysteine-rich metal-binding proteins (e.g. binding Zn2+ and Cd2+) and have been shown to protect against a wide range of stresses including cardiac ischemia-reperfusion and focal cerebral ischemia. To date, the relationship between MT-I + II proteins and anesthetic preconditioning remains to be explored. The studies described in this proposal aim to further our understanding of the molecular mechanisms involved in delayed anesthetic preconditioning in the brain. Specifically in the first study, we will use in vitro mixed cultures of neurons and glia derived from the cortices of wild-type and metallothionein I + II knockout mice to determine the source of protective metallothioneins. Continuing with in vitro culture techniques, we will determine whether MT I + II mediate their neuroprotective effects via extracellular receptors or via intracellular mechanisms. Finally, using confocal microscopy, zinc-sensitive dyes and a FRET-MT construct we will probe the effects of isoflurane on neuronal zinc homeostasis and determine the role of zinc release in the isoflurane-mediated preconditioning pathway. Taken together, these three studies represent an important contribution to understanding mechanisms of delayed anesthetic preconditioning in protecting against ischemic damage. PUBLIC HEALTH RELEVANCE: The proposed research provides both basic knowledge and evaluation in the area of isoflurane preconditioning against ischemic damage. By employing neuronal tissue culture techniques, cell viability assays and confocal microscopy we plan to characterize the role of metallothioneins I and II in mediating anesthetic-mediated protection. This work has important implications for prophylactic treatment against stroke in clinical settings and for operative/perioperative treatment against ischemia.